Biomedical subjects
Publications and source records attributed to J A Lenstra.
The eye lens membrane component 'MP34' [Exp. Eye Res. 24 (1977) 413-415] has been resolved into three protein components and in a revised nomenclature designated MP35, MP36.5 and MP37, respectively. MP37 has been identified as the enzyme glyceraldehyde 3-phosphate dehydrogenase, which is one of the major components of membranes both from cultured hamster lens cells and from HeLa cells.
Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta- or gamma-) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2-3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm-blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non-coding sequences of both vimentin and actin genes are conserved in length.
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Secondary structural elements of the phosphatidylcholine-transfer protein from bovine liver have been predicted from its primary structure with the aid of two computerized methods. The predicted alpha-helix and beta-strand content have been compared with the values derived from circular dichroism spectra. The hydrophobicity profile (Rose plot) of the protein indicated that the supposed lipid-binding site occurs in the most hydrophobic region. The predicted secondary structural elements have been folded in a tentative model of the protein molecule according to its hydrophobicity profile.
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A hybrid RNase S', consisting of synthetic S-peptide of rat ribonuclease and bovine S-protein, was studied by 1H n.m.r. to determine the effects of the many N-terminal amino acid replacements in rat S-peptide as compared to bovine S-peptide. As judged from the aromatic resonances, the conformation of the hybrid RNase S' is essentially identical to the conformation of bovine RNase S'. Notably, the active-site histidines 12 and 119 were not affected by the substitutions in the S-peptide, confirming earlier findings that the catalytic properties of naturally occurring RNases are modulated by the S-protein part of the molecule only. However, the resonances of Tyr-25 and His-48, which in RNase A are involved in a pH-dependent conformational transition, appeared to be different in the hybrid RNase, demonstrating that amino acid replacements may influence the structure of the protein locally.
1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.
The complete amino acid sequence of mouse pancreatic ribonuclease has been determined by analysis of tryptic, chymotryptic, thermolytic and CNBr peptides and by automatic sequence analysis of the intact protein. The sequence of mouse RNase differs in 20--30% of the positions from other RNase sequences. Three unique or neraly unique substitutions were found, viz. Gly-68 leads to Arg-68, Arg-85 leads to His-85 and Ser-123 leads to Thr-123. All these three residues might be involved in interactions with substrate molecules. A most parsimonious tree of the myomorph rodent RNase shows that after the divergence of rat and mouse, the ribonuclease of rat accumulated substitutions at a rate 2.5--4.3 times as high as the rates in other branches of the tree and 23 times as high as the average rate in the Bovidae ribonuclease evolution. These extreme fluctuations in substitution rate are difficult to reconcile with the hypothesis of the evolutionary clock. The high evolution rate of rat ribonuclease is thought to be caused by positive selection, leading to new functional properties of the enzyme.
The amino acid sequence of golden hamster pancreatic ribonuclease was determined by analysis of tryptic, chymotryptic, thermolytic, and CNBr peptides and by automatic sequence analysis of the intact protein. Like all RNases with an Asn-Met-Thr sequence at positions 34-36, hamster RNase is glycosylated at position 34 with a complex-type carbohydrated chain. Val-17, Ala-18, His-55, His-76 and Ala-90 have never been observed in other pancreatic RNases. Ala-90 replaces Ser-90, which had been invariant in all mammalian RNases studied so far. The amino acid sequence of hamster RNase differs at 15 positions from that of another Cricetidae rodent, the muskrat. The similarity between both ribonucleases was used to confirm a few less certain parts of the muskrat RNase sequence. The replacement rate of the RNases of the Cricetidae appeared to be higher than the average rate in the mammals, but much lower than the rate in another myomorph family, the Muridae (mouse and rat). Possibly, in many respects, the Cricetidae underwent less evolutionary change in recent times than the evolutionarily highly successful Muridae.
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Data of 33 proteins are used to compare four methods which predict secondary structure from the amino acid sequence. The prediction of alpha-helices according to the histogram method of Argos et al. (Argos, P., Schwarz, J. and Schwarz, J. (1976) Biochim. Biophys. Acta 439, 261-273) is on the average as reliable as the statistical method of Nagano (Nagano, K. (1973) J. Mol. Biol. 75, 401-420). Both methods predict helices more accurately than the stereo-chemical method of Lim (Lim, V.I. (1974) J. Mol. Biol. 88, 873-894). The method of Nagano yields the best prediction of beta-structure, while the beta-structure predictions of Lim and Argos et al. are not significantly different. The results of the alpha-helix and beta-structure predictions according to the statistical mechanical method of Tanaka and Scheraga (Tanaka, S. and Scheraga, H.A. (1976) Macromolecules 9, 168-182) are inferior to those obtained by the other three methods. For the prediction of turns, no significant difference between the methods of Nagano (Nagano, K. (1974) J. Mol. Biol. 84, 337-372) and Argos et al. was found.
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Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.